Eurycomanone Blocks TGF-β1-Induced Epithelial-to-Mesenchymal Transition, Migration, and Invasion Pathways in Human Non-Small Cell Lung Cancer Cells by Targeting Smad and Non-Smad Signaling

Abstract

Non-small cell lung cancer (NSCLC) is a predominant form of lung cancer that is often diagnosed at an advanced metastatic stage. The processes of cancer cell migration and invasion involve epithelial-to-mesenchymal transition (EMT), which is crucial for metastasis. Targeting cancer aggressiveness with effective plant compounds has gained attention as a potential adjuvant therapy. Eurycomanone (ECN), a bioactive quassinoid found in the root of Eurycoma longifolia Jack, has demonstrated anti-cancer activity against various carcinoma cell lines, including human NSCLC cells. This study aimed to investigate the in vitro effects of ECN on the migration and invasion of human NSCLC cells and to elucidate the mechanisms by which ECN modulates the EMT in these cells. Non-toxic doses (≤IC20) of ECN were determined using the MTT assay on two human NSCLC cell lines: A549 and Calu-1. The results from wound healing and transwell migration assays indicated that ECN significantly suppressed the migration of both TGF-β1-induced A549 and Calu-1 cells. ECN exhibited a strong anti-invasive effect, as its non-toxic doses significantly suppressed the TGF-β1-induced invasion of NSCLC cells through Matrigel and decreased the secretion of MMP-2 from these cancer cells. Furthermore, ECN could affect the TGF-β1-induced EMT process in various ways in NSCLC cells. In TGF-β1-induced A549 cells, ECN significantly restored the expression of E-cadherin by inhibiting the Akt signaling pathway. Conversely, in Calu-1, ECN reduced the aggressive phenotype by decreasing the expression of the mesenchymal protein N-cadherin and inhibiting the TGF-β1/Smad pathway. In conclusion, this study demonstrated the anti-invasive activity of eurycomanone from E. longifolia Jack in human NSCLC cells and provided insights into its mechanism of action by suppressing the effects of TGF-β1 signaling on the EMT program. These findings offer scientific evidence to support the potential of ECN as an alternative therapy for metastatic NSCLC.

Keywords: epithelial-to-mesenchymal transition; eurycomalactone; eurycomanone; invasion; non-small cell lung cancer.

Figure 1

Cytotoxic Effects of ECL and ECN on Human NSCLC Cells. Using an MTT assay. The percentages of cell viability for A549 and Calu-1 were assessed after treatment with varying concentrations of (A,B) ECL (0–20 μM) or (C,D) ECN (0–30 μM) for 24 and 48 h. All data are presented as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 versus the non-treated control.

Figure 2

Effect of ECL and ECN on TGF-β1-Induced Migration in Human NSCLC Cells Using a wound healing assay, the migration of A549 or Calu-1 cells was assessed following co-treatment with or without (A) TGF-β1 (10 ng/mL) + ECL or (B) TGF-β1 (10 ng/mL) + ECN. The percentages of cell migration were normalized to the TGF-β1-treated control group. All data are presented as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 versus the TGF-β1-treated control group. The scale bar = 200 μm.

Figure 3

Effects of ECL and ECN on TGF-β1-induced MMP-2 secretion in human NSCLC Cells. The zymogram of MMP-2 secretion and quantification of MMP-2 secretion level in A549 cells or Calu-1 cells after the co-treatment with or without (A) TGF-β1 (10 ng/mL) + ECL or (B) TGF-β1 (10 ng/mL) + ECN for 24 or 48 h. The intensity of the clear band was normalized against the TGF-β1-treated control group. All data are presented as the mean ± SD of three independent experiments. ** p < 0.01, *** p < 0.001 versus the TGF-β1-treated control group.

Figure 4

Effect of ECN on TGF-β1-induced migration and invasion in human NSCLC cells. The representative images (10× magnification) and quantification of A549 and Calu-1 cells were assessed for (A) cell migration and (B) invasion using transwell migration and invasion assays, respectively. Cells were co-treated with TGF-β1 (10 ng/mL) and ECN for 12, 24, or 48 h. The percentages of migration or invasion were normalized to the TGF-β1-treated control group. All data are presented as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 versus the TGF-β1-treated control group.

Figure 5

Effect of ECN on expression of EMT-associated proteins and TGF-β1 signaling proteins in TGF-β1-treated A549 cells. Representative immunoblots with quantification of (A) E-cadherin, (B) N-cadherin, (C) vimentin, (D) p-Smad2 and total Smad2, and (E) p-Akt and total Akt expression in A549 cells after co-treatment with or without TGF-β1 (10 ng/mL) and ECN for 24 or 48 h. β-actin was used as an internal control. The levels of the targeted proteins were normalized to β-actin and are represented as relative to the TGF-β1-treated control group. All data are presented as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 versus the TGF-β1-treated control group.

Figure 6

Effects of ECN on EMT-associated protein and TGF-β1 signaling protein expression in TGF-β1-treated Calu-1 cells determined by Western blotting. (A) The representative immunoblots with quantification of N-cadherin, (B) vimentin, (C) p-Smad2 and total Smad2 and (D) p-Akt and total Akt expression of Calu-1 cells after co-treated with or without TGF-β1 (10 ng/mL) and ECN for 24 h. β-actin was used as an internal control. The band of targeted protein was normalized with β-actin and represents as relative to the TGF-β1-treated control group. All data are presented as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 versus the TGF-β1-treated control group.